Following an acute cardiovascular accident, the only treatment currently available to recanalize an occluded blood vessel is systemic delivery of a high dose of plasminogen activators. While effective when administered soon after the event, plasminogen activators also cause debilitating side effects such as intracranial haemorrhage and neurotoxicity. In addition, successful restoration of blood flow is not guaranteed because of low recanalization and high reocclusion rates, even when high doses of plasminogen activators are administered [ Saver J L. et al. (2011) J Thromb Haemost. 9 Suppl 1, 333-343]. Accordingly, there remains a need in the art for effective treatments of occluded blood vessels, for example by promoting fibrinolysis or thrombolysis.
One of the causes for thrombolytic failure is the presence of circulating inhibitors of fibrinolysis, such as Thrombin-Activatable Fibrinolysis Inhibitor (TAFT) and plasminogen activator inhibitor 1 (PAI-1) [Fernandez-Cadenas I et al. (2007) J Thromb Haemost. 5, 1862-1868]. Both molecules slow down the tissue type-plasminogen activator (tPA)-mediated formation of plasmin, the key enzyme in fibrinolysis, although through distinct mechanisms (as reviewed in Rijken D C & Lijnen H R (2009) J Thromb Haemost. 7, 4-13). TAFI, a 56 kDa proenzyme with a plasma level of 4-15 μg/ml, can be activated into TAFIa by thrombin, alone or in complex with thrombomodulin, or plasmin. Through its carboxypeptidase activity, TAFIa is able to cleave off C-terminal Lys residues exposed on partially degraded fibrin, which serve as a co-factor function in the tPA-mediated activation of plasminogen into plasmin. PAI-1 (45 kDa glycoprotein with a plasma level of 5-50 ng/ml and a concentration within platelets of 200 ng/ml) is the main inhibitor of tPA and belongs to the serine protease inhibitors (serpin) superfamily. The active form of PAI-1 can irreversibly neutralize the activity of tPA by forming a 1:1 stoichiometric covalent complex, accompanied by deformation of catalytic triad of the serine protease.
Given their complementary roles in inhibiting fibrinolysis, one approach to promoting fibrinolysis is dual inhibition of TAFI and PAI-1. Simultaneous targeting of TAFI and PAI-1 has been attempted in several studies. However, the results did not consistently indicate that dual inhibition of TAFI and PAI-1 improved thrombolysis as compared to single inhibition. In one study, complementary roles of TAFI and PAI-1, as well as a third molecule α2-AP, were characterized in tPA induced thrombolysis assays in the presence or absence of inhibitors of TAFI, PAI-1, and/or α2-AP [Mutch N J. et al. (2007) J Thromb Haemost. 5, 812-817]. Depending on the type of thrombus, the assays indicated either a role for all three molecules or a substantial contribution of α2-AP and TAFI, with a minor contribution from PAI-1. Similarly, single and double knockout studies in mice suggested that thrombolytic effects in certain assays were due to inhibition of TAFI rather than PAI-1 [Vercauteren E et al. (2012) J Thromb Haemost. 10, 2555-2562].
Notably, a dual targeting strategy based on bispecific antibody derivatives (diabodies) has shown promise. The diabody T12D11x33H1F7, based on monoclonal antibodies which bind TAFI and PAI-1, was shown to have a stimulating effect on fibrinolysis which exceeded the effect observed when its component monoclonal antibodies (MA) were tested separately. In addition, new monoclonal antibodies against TAFI and PAI-1 exhibit unique features. MA-RT36A3F5 and MA-TCK26D6 both inhibit mouse and rat TAFI, with each MA acting through distinct mechanisms: the former destabilizes TAFIa, whereas the latter impairs the plasmin-mediated activation of TAFI and also interferes with the interaction of TAFIa on fibrin [Hillmayer K et al. J (2008) Thromb Haemost. 6, 1892-1899; Vercauteren E et al. (2011) Blood 117, 4615-4622; Semeraro F, et al. (2013) J Thromb Haemost. 11, 2137-2147]. MA-33H1F7 and MA-MP2D2 inhibit mouse and rat PAI-1, by converting the active form into a substrate form of PAI-1 which is cleaved by tPA [Debrock S. & Declerck P J. (1997) Biochim Biophys Acta. 1337, 257-266; Van De Craen B. et al. (2011) Thromb Res. 128, 68-76]. In vivo studies have shown a beneficial effect of the above mentioned antibodies on the rate of survival and paralysis in mice after thromboembolic challenge [Vercauteren (2011) cited above, Van De Craen cited above]. Recently, the MA antibodies MA-33H1F7 and MA-TCK26D6 which specifically recognize the corresponding human antigens were adapted to make the bispecific antibody derivative Db-TCK26D6x33H1F7, and a strong profibrinolytic effect of the diabody was demonstrated in vitro [Wyseure T et al. (2013) J Thromb Haemost. 11, 2069-2071]. However, no dual targeting studies to date have conclusively demonstrated a role for inhibitors or diabodies in treating specific thrombotic disorders in vivo. In addition, no studies have provided evidence for viable treatments for thrombotic disorders based on inhibitors of fibrinolysis or thrombolysis as alternatives to plasminogen activators.